The overall goal of this proposal is to understand the mechanism and functional consequence of regulation of the dopamine transporter by a-synuclein. The work described in this application is focused on the problem of whether a-synuclein over-expression affects the dopamine transporter activity, leading to alterations in dopaminergic transmission. The proposed studies will potentially define the molecular mechanisms of dopamine transporter regulation and thus dopaminergic transmission upstream of neuronal injury when a-synuclein is over-expressed. We hypothesize that a-synuclein interacts with the C-terminus domain of the dopamine transporter to alter the ionic permeability of the transporter, thus increasing dopamine efflux and decreasing substrate uptake without an effect on dopamine transporter surface levels. The project will address this hypothesis with the following specific aims: 1) to determine whether a-synuclein modulates the biophysical properties of the dopamine transporter, and therefore its functions such as dopamine transporter-mediated whole cell currents, forward and reverse transport of the substrate, and whether these functions are mediated by alterations in surface levels of the transporter;2) to determine whether a-synuclein regulation of dopamine transporter function is through a physical interaction with the dopamine transporter via a shared binding domain with calcium calmodulin kinase II alpha (CaMKII1) on the C-terminus domain of the dopamine transporter;and 3) to determine the impact of a-synuclein over-expression on dopamine transporter function in human pluripotent cells differentiated to midbrain dopaminergic neurons derived from fibroblasts obtained from Parkinson's disease patients and normal subjects. We will use simultaneous whole cell patch clamp electrophysiology with amperometric quantification of released dopamine via the dopamine transporter, and real-time measurement of substrate uptake to study a-synuclein regulation of the dopamine transporter with high temporal and spatial resolution in primary cultures of mouse midbrain dopaminergic neurons. Using Fluorescence Resonance Energy Transfer, biochemical, and immunoprecipitation strategies, we will determine the regulatory role of a-synuclein over-expression on association of the dopamine transporter with CaMKII1, in addition to the phosphorylation state of the transporter under these conditions. Furthermore, these innovative approaches will be deployed to determine the consequences of a-synuclein regulation of dopamine transporter function in human midbrain dopaminergic neurons obtained from normal human subjects and individuals with idiopathic Parkinson's disease and Parkinson's disease with a-synuclein triplication. As both a-synuclein and the dopamine transporter have been implicated in neurodegenerative diseases such as Parkinson's disease, results from our studies will provide important insight into our mechanistic knowledge of these disease states and could be used to develop novel strategies in disease modeling and targeted drug discovery. PUBLIC HEALTH RELEVANCE: The long-term goal of our studies is to understand the mechanism and functional consequences of regulation of the dopamine transporter (DAT) by a-synuclein. DAT is essential for maintaining the temporal and spatial dimension of dopaminergic transmission, while a-synuclein is involved in dopaminergic transmission through a physical interaction with DAT. Our hypothesis is supported by preliminary data in immortalized DA neurons indicating a-synuclein interaction with the C-terminus domain of DAT regulates dopaminergic transmission by altering the ionic permeability of DAT to increase DAT-mediated DA efflux and decrease DA uptake without an effect on DAT surface distribution that potentially leads to alterations in dopaminergic transmission implicated in neurodegenerative diseases.